Abstract 403: Analysis of RET solvent front mutations shows an effect on protein activity in tumor cells
Authors: Shay Rotkopf, DIkla Haham, Lea Birnbaum, Zohar Barbash, Gabi Tarcic
Journal: Cancer Research (2022)

Abstract The rearranged during transfection (RET) gene encodes a receptor tyrosine kinase that is involved in the activation of several signal transduction pathways. Various RET fusions and point mutations were shown to play a role as oncogene drivers in different cancers. In 2020, two selective RET tyrosine kinase inhibitors (TKIs), pralsetinib and selpercatinib, were approved by the FDA for treatment of RET fusion-positive non-small cell lung cancer, RET-mutant medullary thyroid cancer and RET fusion-positive thyroid cancer. These drugs demonstrate a significant advance in efficacy and safety over previous, multi-targeted TKIs for the treatment of RET-positive patients. However, development of acquired resistance to these agents has been observed through the appearance of on-target secondary solvent front mutations in the RET G810 residue. Moreover, 5 different types of amino acid changes - G810A/C/R/S/V have been identified in this position, of which G810C, G810R and G810S are better characterized but their effect on protein activity is lacking. We used a high-throughput functional assay, in which the mutated protein is expressed together with a fluorescently labeled reporter in a cell-based assay. Using this system, we systematically assessed the activity of different combinations of RET fusions or oncogenic point mutations with one of three different common solvent front mutations and compared them to the activity of the individual mutations. Our results show that when a solvent front mutation is expressed on a RET oncogenic point mutation background, the solvent front mutation consistently decreases the oncogenic protein activity. This is supported by our observation that the individual solvent front mutations expressed on a RET WT background show an activity that is lower than that of the RET WT protein. On a RET fusion background the effect of solvent front mutations is more variable, with some RET fusion-solvent front mutation combinations showing a decreased activity and some not affecting the activity of the fusion protein. Across all mutations and fusions tested, the G810R and G810C mutations had a larger effect on RET activity than that of the G810S mutation. We also compared the effect of drug treatment (pralsetinib and selpercatinib) on RET oncogenic activity in the presence of the different solvent front mutations and observed that on all mutational backgrounds tested, the solvent front mutations have an effect on the response dynamics to the drug, and that the extent of the effect depends on the type of solvent front mutation, with G810R and G810C decreasing sensitivity to drug treatment to a larger extent compared to G810S. Together, our findings provide a better understanding of the differential effects of solvent front mutations on RET activity under different oncogenic contexts. Citation Format: Shay Rotkopf, DIkla Haham, Lea Birnbaum, Zohar Barbash, Gabi Tarcic. Analysis of RET solvent front mutations shows an effect on protein activity in tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 403.